This grant proposes to investigate the interaction of the anti-tubercular drug isoniazid (INH) with the Mycobacterium tuberculosis (Mtb) catalase/peroxidase, KatG. The overall goals are to elucidate the mechanism of INH activation by KatG, and understand the basis by which point mutations in KatG suppress INH activation and give rise to INH resistance in tuberculosis (TB). The first specific aim of this proposal is to study the role of oxyferrous and/or other high-valent iron-oxo intermediates to determine which is the species responsible for isoniazid-activation, using microsecond or faster time-scale techniques such as pulse radiolysis, and laser flash photolysis. The second aim of this research is to obtain structural information regarding the binding of isoniazid to wild-type and mutant KatG proteins, using a combination of X-ray crystallography and traditional biochemical investigations (substrate-binding and reactivity studies). Such studies will lead to an understanding of INH binding and activation on a molecular level, including identifying possible pathways for electron-transfer and hydrogen bonding in KatG. As the final specific aim, in vitro biochemical reactions catalyzed by KatG which lead to INH activation will be explored; these include determining the rate of superoxide consumption by KatG in INH activation, and enzyme assays aimed at measuring the formation of the INH-NAD adduct, which has been found to be a potent inhibitor of InhA (involved in mycolic acid biosynthesis). Thus, the combination of the three specific aims set forth in this research proposal will hopefully unlock the KatG-mediated origin of INH-resistance in multiple drug resistant (MDR) TB. [unreadable] [unreadable]